This invention concerns certain amide derivatives which are useful as inhibitors of cytokine mediated disease. The invention also concerns processes for the manufacture of the amide derivatives of the invention, pharmaceutical compositions containing them and their use in therapeutic methods, for example by virtue of inhibition of cytokine mediated disease.
The amide derivatives disclosed in the present invention are inhibitors of the production of cytokines such as Tumour Necrosis Factor (hereinafter TNF), for example TNFxcex1, and various members of the interleukin (hereinafter IL) family, for example IL-1, IL-6 and IL-8. Accordingly the compounds of the invention will be useful in the treatment of diseases or medical conditions in which excessive production of cytokines occurs, for example excessive production of TNFxcex1 or IL-1. It is known that cytokines are produced by a wide variety of cells such as monocytes and macrophages and that they give rise to a variety of physiological effects which are believed to be important in disease or medical conditions such as inflammation and immunoregulation. For example, TNFxcex1 and IL-1 have been implicated in the cell signalling cascade which is believed to contribute to the pathology of disease states such as inflammatory and allergic diseases and cytokine-induced toxicity. It is also known that, in certain cellular systems, TNFxcex1 production precedes and mediates the production of other cytokines such as IL-1.
Abnormal levels of cytokines have also been implicated in, for example, the production of physiologically-active eicosanoids such as the prostaglandins and leukotrienes, the stimulation of the release of proteolytic enzymes such as collagenase, the activation of the immune system, for example by stimulation of T-helper cells, the activation of osteoclast activity leading to the resorption of calcium, the stimulation of the release of proteoglycans from, for example, cartilage, the stimulation of cell proliferation and to angiogenesis.
Cytokines are also believed to be implicated in the production and development of disease states such as inflammatory and allergic diseases, for example inflammation of the joints (especially rheumatoid arthritis, osteoarthritis and gout), inflammation of the gastrointestinal tract (especially inflammatory bowel disease, ulcerative colitis, Crohn""s disease and gastritis), skin disease (especially psoriasis eczema and dermatitis) and respiratory disease (especially asthma, bronchitis, allergic rhinitis and adult respiratory distress syndrome), and in the production and development of various cardiovascular and cerebrovascular disorders such as congestive heart failure, myocardial infarction. the formation of atherosclerotic plaques, hypertension, platelet aggregation, angina, stroke, reperfusion injury, vascular injury including restenosis and peripheral vascular disease, and, for example, various disorders of bone metabolism such as osteoporosis (including senile and postmenopausal osteoporosis). Paget""s disease, bone metastases, hypercalcaemia, hyperparathyroidism, osteosclerosis, osteoperosis and periodontitis, and the abnormal changes in bone metabolism which may accompany rheumatoid arthritis and osteoarthritis. Excessive cytokine production has also been implicated in mediating certain complications of bacterial, fungal and/or viral infections such as endotoxic shock, septic shock and toxic shock syndrome and in mediating certain complications of CNS surgery or injury such as neurotrauma and ischaemic stroke. Excessive cytokine production has also been implicated in mediating or exacerbating the development of diseases involving cartilage or muscle resorption, pulmonary fibrosis, cirrhosis, renal fibrosis, the cachexia found in certain chronic diseases such as malignant disease and acquired immune deficiency syndrome (AIDS), tumour invasiveness and tumour metastasis and multiple sclerosis.
Evidence of the central role played by TNFxcex1 in the cell signalling cascade which gives rise to rheumatoid arthritis is provided by the efficacy in clinical studies of antibodies of TNFxcex1 (The Lancet, 1994, 344, 1125 and British Journal of Rheumatology, 1995, 34, 334).
Thus cytokines such as TNFxcex1 and IL-1 are believed to be important mediators of a considerable range of diseases and medical conditions. Accordingly it is expected that inhibition of the production of and/or effects of these cytokines will be of benefit in the prophylaxis, control or treatment of such diseases and medical conditions.
Without wishing to imply that the compounds disclosed in the present invention possess pharmacological activity only by virtue of an effect on a single biological process, it is believed that the compounds inhibit the effects of cytokines by virtue of inhibition of the enzyme p38 kinase. p38 kinase, otherwise known as cytokine suppressive binding protein (hereinafter CSBP) and reactivating kinase (hereinafter RK), is a member of the mitogen-activated protein (hereinafter MAP) kinase family of enzymes which is known to be activated by physiological stress such as that induced by ionising radiation, cytotoxic agents, and toxins, for example endotoxins such as bacterial lipopolysaccharide, and by a variety of agents such as the cytokines, for example TNFxcex1 and IL-1. It is known that p38 kinase phosphorylates certain intracellular proteins which are involved in the cascade of enzymatic steps which leads to the biosynthesis and excretion of cytokines such as TNFa and IL-1. Known inhibitors of p38 kinase have been reviewed by G. J. Hanson in Expert Opinions on Therapeutic Patents, 1997, 7, 729-733. p38 kinase is known to exist in isoforms identified as p38xcex1 and p38xcex2.
The compounds disclosed in the present invention are inhibitors of the production of cytokines such as TNF, in particular of TNFxcex1, and various interleukins, in particular IL-1.
It is known from J. Med. Chem., 1996, 39, 3343-3356, that certain benzamide derivatives can upregulate the expression of the low density lipoprotein (LDL) receptor in human hepatocyte cells. The disclosed compounds included certain N-(2-methoxyphenyl)- and N-(2-halogenophenyl)-benzamide derivatives.
The compound N-(5-benzamido-2-chlorophenyl)benzamide is disclosed in J. Chem. Res. Synop., 1998, 182-183, 886-896 (Chemical Abstracts volume 129, abstract 67538).
According to one aspect of the present invention there is provided a compound of the Formula I 
wherein:
R1 and R2, which may be the same or different, are selected from hydroxy, C1-6alkoxy, mercapto, C1-6alkylthio, amino, C1-6alkylamino, di-(C1-6alkyl)amino, carboxy, C1-6alkoxycarbonyl, carbamoyl, C1-6alkylcarbamoyl, di-C1-6alkylcarbamoyl, C1-6alkylsulphinyl, C1-6alkylsulphonyl, arylsulphinyl, arylsulphonyl, C1-6alkylaminosulphonyl, di-(C1-6alkyl)aminosulphonyl, nitro, cyano, cyanoC1-6alkyl, hydroxyC1-6alkyl, aminoC1-6alkyl, C1-6alkanoylamino, C1-6alkoxycarbonylamino, C1-6alkanoyl, C1-6alkanoyloxy, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, halo, trifluoromethyl, aryl, arylC1-6alkyl, arylC1-6alkoxy, heteroaryl, heteroarylC1-6alkyl, heterocyclyl and heterocyclyC1-6alkyl;
m and p are independently 0-3, and when m and/or p is 2 or 3 each R1 or R2 group may be the same or different;
R3 is halo, cyano or C1-6alkoxy;
q is 0-4; and
R4 is aryl or cycloalkyl wherein R4 is optionally substituted with up to 3 substituents having any value defined for each R1 group;
or a pharmaceutically-acceptable salt or in-vivo-cleavable ester thereof;
with the proviso that:
N-[5-(3-cyclohexylpropionylamnino)-2-methoxyphenyl]-4-acetoxybenzamide,
N-[2-bromo-5-(3-cyclohexylpropionylamino)phenyl]-4-hydroxybenzamide,
N-[2-chloro-5-(3-cyclohexylpropionylamino)phenyl]-4-acetoxybenzamide,
N-[2-chloro-5-(3-cyclohexylpropionylamino)phenyl]-4-hydroxybenzamide,
N-[2-fluoro-5-(3-cyclohexylpropionylamino)phenyl]-4-hydroxybenzamide and
N -(5-benzamido-2-chlorophenyl)benzamide
are excluded.
xe2x80x9cArylxe2x80x9d in terms such as xe2x80x9carylxe2x80x9d, xe2x80x9carylC1-6alkylxe2x80x9d, xe2x80x9carylthioxe2x80x9d, xe2x80x9carylsulphinylxe2x80x9d, xe2x80x9carylsulphonylxe2x80x9d and xe2x80x9carylC1-6alkoxyxe2x80x9d typically means phenyl or naphthyl, preferably phenyl. xe2x80x9cHeteroarylxe2x80x9d in the terms xe2x80x9cheteroarylxe2x80x9d and xe2x80x9cheteroarylC1-6alkylxe2x80x9d means an aromatic mono- or bicyclic 5-10 membered ring with up to five ring heteroatoms selected from nitrogen, oxygen and sulphur. Examples of xe2x80x98heteroarylxe2x80x99 include thienyl, pyrrolyl, furyl, imidazolyl, oxazolyl, thiazolyl, pyrazolyl, isoxazolyl, isothiazolyl, pyrazinyl, pyridazinyl, pyrimidinyl, pyridyl, indolyl, benzofuranyl, benzothienyl, benzimidazolyl, benzothiazolyl, quinolyl, isoquinolyl, quinazolinyl, quinoxalinyl and cinnolinyl. xe2x80x9cHeterocyclylxe2x80x9d in the terms xe2x80x9cheterocyclylxe2x80x9d and xe2x80x9cheterocyclylC1-6alkylxe2x80x9d means a non-aromatic mono- or bicyclic 5-10 membered ring with up to five ring hetero atoms selected from nitrogen, oxygen and sulphur. Examples of xe2x80x98heterocyclylxe2x80x99 include pyrrolinyl, pyrrolidinyl, morpholinyl, piperidinyl, homopiperidinyl, piperazinyl, homopiperazinyl, dihydropyridinyl and dihydropyrimidinyl. xe2x80x9cCycloalkylxe2x80x9d means a non-aromatic mono- or bicyclic 5-10 membered carbon ring. Examples of xe2x80x9ccycloalkylxe2x80x9d include cyclopentyl, cyclohexyl, cycloheptyl, bicyclo[2.2.1]heptyl and bicyclo[4.4.0]decyl.
Typical values for other generic groups include: for C1-6alkoxy, for example, methoxy and ethoxy, for C1-6alkylthio, for example, methylthio and ethylthio, for C1-6alkylamino, for example, methylamino and ethylamino, for di-(C1-6alkyl)amino, for example, dimethylamino, for C1-6alkoxycarbonyl, for example, methoxycarbonyl and ethoxycarbonyl, for C1-6alkylcarbamoyl, for example, methylcarbamoyl, for di-C1-6alkylcarbamoyl, for example, dimethylcarbamoyl, for C1-6alkylsulphinyl, for example, methylsulphinyl, for C1-6alkylsulphonyl, for example, methylsulphonyl, for C1-6alkylaminosulphonyl, for example, methylaminosulphonyl, for di-(C1-6alkyl)aminosulphonyl, for example, dimethylaminosulphonyl, for cyanoC1-6alkyl, for example, cyanomethyl, for hydroxyC1-6alkyl, for example, hydroxymethyl, for aminoC1-6alkyl, for example, aminomethyl, for C1-6alkanoylamino, for example, formamido and acetamido, for C1-6alkoxycarbonylamino, for example, methoxycarbonylamino, for C1-6alkanoyl, for example, formyl and acetyl, for C1-6alkanoyloxy, for example, acetoxy, for C1-6alkyl, for example, methyl, ethyl, isopropyl and tert-butyl, for C2-6alkenyl, for example, vinyl and allyl, for C2-6alkynyl, for example, ethynyl and 2-propynyl, for halo, for example, fluoro, chloro and bromo, for arylC1-6alkyl, for example, benzyl, and for arylC1-6alkoxy, for example, benzyloxy.
Any ring in R1 or R2 or any ring in a substituent on R4 may be optionally substituted, for example by up to 3 substituents. Suitable substituents include: hydroxy, C1-6alkoxy, mercapto, C1-6alkylthio, amino, C1-6alkylamino, di-(C1-6alkyl)amino, carboxy, carbamoyl, C1-6alkylcarbamoyl, di-C1-6alkylcarbamoyl, C1-6alkylsulphinyl, C1-6alkylsulphonyl, arylsulphinyl, arylsulphonyl, C1-6alkylaminosulphonyl, di-(C1-6alkyl)aminosulphonyl, nitro, cyano, cyanoC1-6alkyl, hydroxyC1-6alkyl, amninoC1-6alkyl, C1-6alkanoylamino, C1-6alkoxycarbonylamino, C1-6alkanoyl, C1-6alkanoyloxy, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, halo and trifluoromethyl.
In this specification the generic term xe2x80x9calkylxe2x80x9d includes both straight-chain and branched-chain alkyl groups. However references to individual alkyl groups such as xe2x80x9cpropylxe2x80x9d are specific for the straight-chain version only and references to individual branched-chain alkyl groups such as xe2x80x9cisopropylxe2x80x9d are specific for the branched-chain version only. An analogous convention applies to other generic terms.
It is to be understood that, insofar as certain of the compounds of Formula I defined above may exist in optically active or racemic forms by virtue of one or more asymmetric carbon atoms, the invention includes in its definition any such optically active or racemic form which possesses the property of inhibiting cytokines, in particular TNF. The synthesis of optically active forms may be carried out by standard techniques of organic chemistry well known in the art, for example by synthesis from optically active starting materials or by resolution of a racemic form. Similarly, inhibitory properties against TNF may be evaluated using the standard laboratory techniques referred to hereinafter.
Preferably R1 is hydroxy, C1-6alkoxy, amino, C1-6alkylamino, di-(C1-6alkyl)amino, carboxy, C1-6alkoxycarbonyl, carbamoyl, C1-6alkylcarbarnoyl, cyano, C1-6alkanoylamino, C1-6alkanoyl, C1-6alkanoyloxy, C1-6alkyl, halo, trifluoromethyl or heterocyclyl. Further preferably R1 is aminoC1-6alkyl.
More preferably R1 is hydroxy, C1-6alkoxy, cyano, halo, morpholino or 4-methylpiperazin-1-yl.
Preferably m is 1 or 2.
Conveniently p is 1 and R2 is C1-6alkoxy, carboxy, C1-6alkoxycarbonyl, C1-6alkyl or halo.
Preferably p is 0.
Preferably R3 is halo.
Preferably q is 0, 1 or 2. More preferably q is 0.
Preferably R4 is phenyl, cyclohexyl or cyclopentyl.
More preferably R4 is phenyl.
Preferred substituents on R4 are hydroxy, C1-6alkoxy, amino, C1-6alkylamino, di-(C1-6alkyl)amino, carboxy, C1-6alkoxycarbonyl, nitro, cyano, C1-6alkanoylamino, C1-6alkanoyl, C1-6alkanoyloxy, C1-6alkyl, halo, trifluoromethyl, phenyl, phenylC1-6alkoxy and heterocyclyl.
More preferably substituents on R4 are selected from hydroxy, cyano, dimethylamino, methoxy, ethoxy, fluoro, chloro and morpholino.
Particular novel compounds of the invention include, for example, amide derivatives of the Formula I, or pharnaceutically-acceptable salts thereof, subject to the exclusions defined hereinbefore, wherein:
(a) R1 is hydroxy, C1-6alkoxy, amino, C1-6alkylamino, di-(C1-6alkyl)amino, carboxy, C1-6alkoxycarbonyl, nitro, cyano, cyanoC1-6alkyl, hydroxyC1-6alkyl, aminoC1-6alkyl, C1-6alkanoylamino, C1-6alkoxycarbonylamino, C1-6alkanoyl, C1-6alkanoyloxy, C1-6alkyl, halo or trifluoromethyl, and m is 1 or 2; and R2, R3, R4, p and q have any of the meanings defined hereinbefore or in this section relating to particular novel compounds of the invention;
(b) R1 is a non-aromatic saturated 5- or 6-membered heterocyclic ring with one or two heteroatoms selected from nitrogen, oxygen and sulphur, and m is 1 or 2; and R2, R3, R4, p and q have any of the meanings defined hereinbefore or in this section relating to particular novel compounds of the invention;
(c) R1 is a saturated heterocyclic ring selected from pyrrolidinyl, morpholinyl, piperidinyl, piperazinyl and 4-(C1-6alkyl)piperazinyl, and m is 1 or 2; and R2, R3, R4, p and q have any of the meanings defined hereinbefore or in this section relating to particular novel compounds of the invention;
(d) R2 is hydroxy, C1-6alkoxy, amino, C1-6alkylamino, di-(C1-6alkyl)amino, carboxy, C1-6alkoxycarbonyl, nitro, cyano, C1-6alkyl, halo or trifluoromethyl, and p is 1; and R1, R3, R4, m and q have any of the meanings defined hereinbefore or in this section relating to particular novel compounds of the invention;
(e) p is 0; and R1, R3, R4, m and q have any of the meanings defined hereinbefore or in this section relating to particular novel compounds of the invention;
(f) R3 is halo; and R1, R2, R4, m, p and q have any of the meanings defined hereinbefore or in this section relating to particular novel compounds of the invention;
(g) q is 1, 2, 3 or 4, and R4 is cycloalkyl; and R1, R2, R3, m and p have any of the meanings defined hereinbefore or in this section relating to particular novel compounds of the invention;
(h) q is 0, and R4 is phenyl which is optionally substituted with up to 3 substituents selected from hydroxy, C1-6alkoxy, amino, C1-6alkylamino, di-(C1-6alkyl)amino, carboxy, C1-6alkoxycarbonyl, nitro, cyano, C1-6alkoxycarbonylamino, C1-6alkanoyl, C1-6alkanoyloxy, C1-6alkyl, halo, trifluoromethyl, phenyl, benzyl and benzyloxy; and R1, R2, R3, m and p have any of the meanings defined hereinbefore or in this section relating to particular novel compounds of the invention;
(i) q is 0, and R4 is phenyl which is substituted with 1 or 2 substituents selected from heteroaryl, heteroarylC1-6alkyl, heterocyclyl and heterocyclylC1-6alkyl; and R1, R2, R3, m and p have any of the meanings defined hereinbefore or in this section relating to particular novel compounds of the invention;
(j) q is 0, and R4 is phenyl which is substituted with 1 or 2 heterocyclyl groups comprising a non-aromatic saturated 5- or 6-membered heterocyclic ring with one or two heteroatoms selected from nitrogen, oxygen and sulphur; and R1, R2, R3, m and p have any of the meanings defined hereinbefore or in this section relating to particular novel compounds of the invention; and
(k) q is 0, and R4 is phenyl which is substituted with 1 or 2 heterocyclic groups selected from pyrrolidinyl, morpholinyl, piperidinyl, piperazinyl and 4-(C1-6alkyl)piperazinyl; and R1, R2, R3, m and p have any of the meanings defined hereinbefore or in this section relating to particular novel compounds of the invention.
A preferred compound of the invention is an amide derivative of the Formula I wherein R1 is hydroxy, methoxy, ethoxy, propoxy, isopropoxy, butoxy, amino, methylamino, ethylamino, dimethylamino, diethylamino, carboxy, methoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl, cyano, acetamido, acetyl, acetoxy, methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, fluoro, chloro, trifluoromethyl, pyrrolidin-1-yl, morpholino, piperidino, piperazin-1-yl or 4-methylpiperazin-1-yl;
m is 1 or 2;
p is 0;
R3 is fluoro, chloro or bromo;
q is 1, 2 or 3; and
R4 is cyclohexyl or cyclopentyl;
or a pharmaceutically-acceptable salt thereof.
A further preferred compound of the invention is an amide derivative of the Formula I wherein R1 is hydroxy, methoxy, ethoxy, propoxy, isopropoxy, butoxy, amino, methylamino, ethylamino, dimethylamino, diethylamino, carboxy, methoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl, cyano, acetamido, acetyl, acetoxy, methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, fluoro, chloro, trifluoromethyl, pyrrolidin-1-yl, morpholino, piperidino, piperazin-1-yl or 4-methylpiperazin-1-yl;
m is 1 or 2;
p is 0;
R3 is fluoro, chloro or bromo;
q is 0; and
R4 is phenyl which is optionally substituted with 1 or 2 substituents selected from hydroxy, methoxy, ethoxy, propoxy, amino, methylamino, ethylamino, propylamino, dimethylamino, diethylamino, carboxy, methoxycarbonyl, ethoxycarbonyl, nitro, cyano, acetamido, acetyl, acetoxy, methyl, ethyl, fluoro, chloro, bromo, trifluoromethyl, phenyl, benzyloxy, pyrrolidin-1-yl, morpholino, piperidino, piperazin-1-yl and 4-methylpiperazin-1-yl;
or a pharmaceutically-acceptable salt thereof.
A further preferred compound of the invention is an amide derivative of the Formula I wherein R1 is hydroxy, methoxy, ethoxy, cyano, fluoro, chloro, morpholino or 4-methylpiperazin-1-yl;
m is 1 or 2;
p is 0;
R3 is fluoro, chloro or bromo;
q is 0; and
R4 is phenyl which is substituted with 1 or 2 substituents selected from hydroxy, methoxy, dimethylamino, methoxycarbonyl, cyano, fluoro, chloro and morpholino;
or a pharmaceutically-acceptable salt thereof.
A further preferred compound of the invention is an amide derivative of the Formula I wherein R1 is hydroxy, methoxy, ethoxy, amino, cyano, acetoxy, fluoro, chloro, morpholino or 4-methylpiperazin-1-yl;
m is 1 or 2;
p is 0;
R3 is fluoro, chloro or bromo;
q is 0; and
R4 is phenyl which is unsubstituted or substituted with 1 or 2 substituents selected from hydroxy, methoxy, amino, dimethylamino, methoxycarbonyl, nitro, cyano, fluoro, chloro and morpholino;
or a pharmaceutically-acceptable salt thereof.
Particular preferred compounds of the invention include, for example:
N-[2-chloro-5-(3-cyanobenzamido)phenyl]-3,4-dimethoxybenzamide,
N-[2-chloro-5-(3-dimethylaminobenzamido)phenyl]-3,4-dimethoxybenzamide,
N-[2chloro-5-(4-cyanobenzamido)phenyl]-3,4-dimethoxybenzamide and
N-[2-chloro-5-(4-cyanobenzamido)phenyl]-3-(4-methylpiperazin-1-yl)benzamide;
or the pharmaceutically-acceptable salts thereof.
Further particular preferred compounds of the invention include, for example:
N-(5-benzamido-2-chlorophenyl)-3,4-dimethoxybenzamide,
N-[2-chloro-5-(3-morpholinobenzamido)phenyl]-3,4-dimethoxybenzamide,
N-[5-(4-acetoxybenzamido)-2-chlorophenyl]-4-cyanobenzamide,
N-(5-benzamido-2-chlorophenyl)-2-amino-4-methoxybenzamide and
N-[2-chloro-5-(3-morpholinobenzamido)phenyl]-4-cyanobenzamide;
or the pharmaceutically-acceptable salts thereof.
A suitable pharmaceutically-acceptable salt of a compound of the Formula I is, for example, an acid-addition salt of a compound of the Formula I which is sufficiently basic, for example an acid-addition salt with an inorganic or organic acid such as hydrochloric, hydrobromic, sulphuric, trifluoroacetic, citric or maleic acid; or, for example a salt of a compound of the Formula I which is sufficiently acidic, for example an alkali or alkaline earth metal salt such as a calcium or magnesium salt, or an ammonium salt, or a salt with an organic base such as methylamine, dimethylamine, trimethylamine, piperidine, morpholine or tris-(2-hydroxyethyl)amine.
Various forms of prodrugs are known in the art. For examples of such prodrug derivatives. see:
a) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985) and Methods in Enzymology, Vol. 42, p. 309-396, edited by K. Widder, et al. (Academic Press, 1985);
b) A Textbook of Drug Design and Development, edited by Krogsgaard-Larsen and H. Bundgaard, Chapter 5 xe2x80x9cDesign and Application of Prodrugsxe2x80x9d, by H. Bundgaard p. 113-191 (1991);
c) H. Bundgaard, Advanced Drug Delivery Reviews, 8, 1-38 (1992);
d) H. Bundgaard, et al., Journal of Pharmaceutical Sciences, 77, 285 (1988); and
e) N. Kakeya, et al., Chem Pharm Bull, 32,692 (1984).
Examples of such pro-drugs may be used to form in-vivo-cleavable esters of a compound of the Formula I. An in-vivo-cleavable ester of a compound of the Formula I containing a carboxy group is, for example, a pharmaceutically-acceptable ester which is cleaved in the human or animal body to produce the parent acid. Suitable pharmaceutically-acceptable esters for carboxy include C1-6alkoxymethyl esters, for example methoxymethyl; C1-6alkanoyloxymethyl esters, for example pivaloyloxymethyl; phthalidyl esters; C3-8cycloalkoxycarbonyloxyC1-6alkyl esters, for example 1-cyclohexylcarbonyloxyethyl; 1,3-dioxolan-2-ylmethyl esters, for example 5-methyl-1,3-dioxolan-2-ylmethyl; and C1-6alkoxycarbonyloxyethyl esters, for example 1-methoxycarbonyloxyethyl; and may be formed at any carboxy group in the compounds of this invention.
In order to use a compound of the Formula I or a pharmaceutically acceptable salt or in-vivo-cleavable ester thereof for the therapeutic treatment (including prophylactic treatment) of mammals including humans, it is normally formulated in accordance with standard pharmaceutical practice as a pharmaceutical composition.
According to this aspect of the invention there is provided a pharmaceutical composition which comprises an amide derivative of the Formula I, or a pharmaceutically-acceptable salt or in-vivo-cleavable ester thereof, as defined hereinbefore in association with a pharmaceutically-acceptable diluent or carrier.
The compositions of the invention may be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), for topical use (for example as creams, ointments, gels, or aqueous or oily solutions or suspensions), for administration by inhalation (for example as a finely divided powder or a liquid aerosol), for administration by insufflation (for example as a finely divided powder) or for parenteral administration (for example as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular or intramuscular dosing or as a suppository for rectal dosing).
The compositions of the invention may be obtained by conventional procedures using conventional pharmaceutical excipients, well known in the art. Thus, compositions intended for oral use may contain, for example, one or more colouring, sweetening, flavouring and/or preservative agents.
Suitable pharmaceutically-acceptable excipients for a tablet formulation include, for example, inert diluents such as lactose, sodium carbonate, calcium phosphate or calcium carbonate, granulating and disintegrating agents such as corn starch or algenic acid: binding agents such as starch; lubricating agents such as magnesium stearate stearic acid or talc; preservative agents such as ethyl or propyl p-hydroxybenzoate, and anti-oxidants, such as ascorbic acid. Tablet formulations may be uncoated or coated either to modify their disintegration and the subsequent absorption of the active ingredient within the gastrointestinal tract, or to improve their stability and/or appearance, in either case, using conventional coating agents and procedures well known in the art.
Compositions for oral use may be in the form of hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, for example, calciun carbonate, calcium phosphate or kaolin, or as soft gelatin capsules in which the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin, or olive oil.
Aqueous suspensions generally contain the active ingredient in finely powdered form together with one or more suspending agents. such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia: dispersing or wetting agents such as lecithin or condensation products of an alkylene oxide with fatty acids (for example polyoxethylene stearate), or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives (such as ethyl or propyl p-hydroxybenzoate, anti-oxidants (such as ascorbic acid), colouring agents, flavouring agents, and/or sweetening agents (such as sucrose, saccharine or aspartame).
Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil (such as arachis oil, olive oil, sesame oil or coconut oil) or in a mineral oil (such as liquid paraffin). The oily suspensions may also contain a thickening agent such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set out above, and flavouring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water generally contain the active ingredient together with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients such as sweetening, flavouring and colouring agents, may also be present.
The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil. such as olive oil or arachis oil, or a mineral oil, such as for example liquid paraffin or a mixture of any of these. Suitable emulsifying agents may be, for example, naturally-occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as soya bean, lecithin, an esters or partial esters derived from fatty acids and hexitol anhydrides (for example sorbitan monooleate) and condensation products of the said partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening, flavouring and preservative agents.
Syrups and elixirs may be formulated with sweetening agents such as glycerol, propylene glycol, sorbitol, aspartame or sucrose, and may also contain a demulcent, preservative, flavouring and/or colouring agent.
The pharmaceutical compositions may also be in the form of a sterile injectable aqueous or oily suspension, which may be formulated according to known procedures using one or more of the appropriate dispersing or wetting agents and suspending agents, which have been mentioned above. A sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example a solution in 1,3-butanediol.
Suppository formulations may be prepared by mixing the active ingredient with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Suitable excipients include, for example, cocoa butter and polyethylene glycols.
Topical formulations, such as creams, ointments, gels and aqueous or oily solutions or suspensions, may generally be obtained by formulating an active ingredient with a conventional, topically acceptable, vehicle or diluent using conventional procedures well known in the art.
Compositions for administration by insufflation may be in the form of a finely divided powder containing particles of average diameter of, for example, 30 xcexcm or much less, the powder itself comprising either active ingredient alone or diluted with one or more physiologically acceptable carriers such as lactose. The powder for insufflation is then conveniently retained in a capsule containing, for example, 1 to 50 mg of active ingredient for use with a turbo-inhaler device, such as is used for insufflation of the known agent sodium cromoglycate.
Compositions for administration by inhalation may be in the form of a conventional pressurised aerosol arranged to dispense the active ingredient either as an aerosol containing finely divided solid or liquid droplets. Conventional aerosol propellants such as volatile fluorinated hydrocarbons or hydrocarbons may be used and the aerosol device is conveniently arranged to dispense a metered quantity of active ingredient.
For further information on Formulation the reader is referred to Chapter 25.2 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990.
The amount of active ingredient that is combined with one or more excipients to produce a single dosage form will necessarily vary depending upon the host treated and the particular route of administration. For example, a formulation intended for oral administration to humans will generally contain, for example, from 0.5 mg to 2 g of active agent compounded with an appropriate and convenient amount of excipients which may vary from about 5 to about 98 percent by weight of the total composition. Dosage unit forms will generally contain about 1 mg to about 500 mg of an active ingredient. For further information on Routes of Administration and Dosage Regimes the reader is referred to Chapter 25.3 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990.
The size of the dose for therapeutic or prophylactic purposes of a compound of the Formula I will naturally vary according to the nature and severity of the conditions, the age and sex of the animal or patient and the route of administration, according to well known principles of medicine.
In using a compound of the Formula I for therapeutic or prophylactic purposes it will generally be administered so that a daily dose in the range, for example, 0.5 mg to 75 mg per kg body weight is received, given if required in divided doses. In general lower doses will be administered when a parenteral route is employed. Thus, for example, for intravenous administration, a dose in the range, for example, 0.5 mg to 30 mg per kg body weight will generally be used. Similarly, for administration by inhalation, a dose in the range, for example, 0.5 mg to 25 mg per kg body weight will be used. Oral administration is however preferred, particularly in tablet form. Typically, unit dosage forms will contain about 1 mg to 500 mg of a compound of this invention.
According to a further aspect of the invention there is provided an amide derivative of the Formula I, or a pharmaceutically-acceptable salt or in-vivo-cleavable ester thereof, as defined hereinbefore for use in a method of treatment of the human or animal body by therapy.
According to a further aspect of the invention there is provided the use of an amide derivative of the Formula I, or a pharmaceutically-acceptable salt or in-vivo-cleavable ester thereof, as defined hereinbefore or any of those known compounds excluded from the definition of the compounds of the invention in the manufacture of a medicament for use in the treatment of diseases or medical conditions mediated by cytokines.
In a further aspect the present invention provides a method of treating diseases or medical conditions mediated by cytokines which comprises administering to a warm-blooded animal an effective amount of a compound of the Formula I, or a pharmaceutically-acceptable salt or in-vivo-cleavable ester thereof.
In a further aspect the present invention provides the use of a compound of the Formula I, or a pharmaceutically-acceptable salt or in-vivo-cleavable ester thereof, in the manufacture of a medicament for use in the treatment of diseases or medical conditions mediated by TNF, IL-1, IL-6 or IL-8.
In a further aspect the present invention provides a method of treating diseases or medical conditions mediated by TNF, IL-1, IL-6 or IL-8 which comprises administering to a warm-blooded animal an effective amount of a compound of the Formula I, or a pharmaceutically-acceptable salt or in-vivo-cleavable ester thereof.
In a further aspect the present invention provides the use of a compound of the Formula I, or a pharmaceutically-acceptable salt or in-vivo-cleavable ester thereof in the manufacture of a medicament for use in the treatment of diseases or medical conditions mediated by TNF.
In a further aspect the present invention provides a method of treating diseases or medical conditions mediated by TNF which comprises administering to a warm-blooded animal an effective amount of a compound of the Formula I, or a pharmaceutically-acceptable salt or in-vivo-cleavable ester thereof.
In a further aspect the present invention provides the use of a compound of the Formula I, or a pharmaceutically-acceptable salt or in-vivo-cleavable ester thereof, in the manufacture of a medicament for use in inhibiting TNF, IL-1, IL-6 or IL-8.
In a further aspect the present invention provides a method of inhibiting TNF, IL-1, IL-6 or IL-8 which comprises administering to a warm-blooded animal an effective amount of a compound of the Formula I, or a pharmaceutically-acceptable salt or in-vivo-cleavable ester thereof.
In a further aspect the present invention provides the use of a compound of the Formula I, or a pharmaceutically-acceptable salt or in-vivo-cleavable ester thereof, in the manufacture of a medicament for use in inhibiting TNF.
In a further aspect the present invention provides a method of inhibiting TNF which comprises administering to a warm-blooded animal an effective amount of a compound of the Formula I, or a pharmaceutically-acceptable salt or in-vivo-cleavable ester thereof.
In a further aspect the present invention provides the use of a compound of the Formula I, or a pharmaceutically-acceptable salt or in-vivo-cleavable ester thereof in the manufacture of a medicament for use in the treatment of diseases or medical conditions mediated by p38 kinase.
In a further aspect the present invention provides a method of treating diseases or medical conditions mediated by p38 kinase which comprises administering to a warm-blooded animal an effective amount of a compound of the Formula I, or a pharmaceutically-acceptable salt or in-vivo-cleavable ester thereof.
In a further aspect the present invention provides the use of a compound of the Formula I, or a pharmaceutically-acceptable salt or in-vivo-cleavable ester thereof. in the manufacture of a medicament for use in the production of a p38 kinase inhibitory effect.
In a further aspect the present invention provides a method of providing a p38 kinase inhibitory effect which comprises administering to a warm-blooded animal an effective amount of a compound of the Formula I, or a pharmaceutically-acceptable salt or in-vivo-cleavable ester thereof.
In a further aspect the present invention provides the use of a compound of the Formula I, or a pharmaceutically-acceptable salt or in-vivo-cleavable ester thereof, in the manufacture of a medicament for use in the treatment of rheumatoid arthritis, asthma, irritable bowel disease, multiple sclerosis, AIDS, septic shock, ischaemic heart disease or psoriasis.
In a further aspect the present invention provides a method of treating rheumatoid arthritis, asthma, irritable bowel disease, multiple sclerosis, AIDS, septic shock, ischaemic heart disease or psoriasis which comprises administering to a warm-blooded animal an effective amount of a compound of the Formula I, or a pharmaceutically-acceptable salt or in-vivo-cleavable ester thereof.
The compounds of this invention may be used in combination with other drugs and therapies used in the treatment of disease states which would benefit from the inhibition of cytokines, in particular TNF and IL-1. For example, the compounds of the Formula I could be used in combination with drugs and therapies used in the treatment of rheumatoid arthritis, asthma, irritable bowel disease, multiple sclerosis, AIDS, septic shock, ischaemic heart disease, psoriasis and the other disease states mentioned earlier in this specification.
For example, by virtue of their ability to inhibit cytokines, the compounds of the Formula I are of value in the treatment of certain inflammatory and non-inflammatory diseases which are currently treated with a cyclooxygenase-inhibitory non-steroidal anti-inflammatory drug (NSAID) such as indomethacin, ketorolac, acetylsalicyclic acid, ibuprofen, sulindac, tolmetin and piroxicam. Co-administration of a compound of the Formula I with a NSAID can result in a reduction of the quantity of the latter agent needed to produce a therapeutic effect. Thereby the likelihood of adverse side-effects from the NSAID such as gastrointestinal effects are reduced. Thus according to a further feature of the invention there is provided a pharmaceutical composition which comprises a compound of the Formula I, or a pharnaceutically-acceptable salt or in-vivo-cleavable ester thereof, in conjunction or admixture with a cyclooxygenase inhibitory non-steroidal anti-inflammatory agent, and a pharmaceutically-acceptable diluent or carrier.
The compounds of the invention may also be used with anti-inflammatory agents such as an inhibitor of the enzyme 5-lipoxygenase (such as those disclosed in European Patent Applications Nos. 0351194, 0375368. 0375404, 0375452, 037547, 0381375, 0385662, 0385663, 0385679, 0385680).
The compounds of the Formula I may also be used in the treatment of conditions such as rheumatoid arthritis in combination with antiarthritic agents such as gold, methotrexate, steroids and penicillinamine, and in conditions such as osteoarthritis in combination with steroids.
The compounds of the present invention may also be administered in degradative diseases, for example osteoarthritis, with chondroprotective, anti-degradative and/or reparative agents such as Diacerhein, hyaluronic acid formulations such as Hyalan. Rumalon, Arteparon and glucosamine salts such as Antril.
The compounds of the Formula I may be be used in the treatment of asthma in combination with antiasthmatic agents such as bronchodilators and leukotriene antagonists.
If formulated as a fixed dose such combination products employ the compounds of this invention within the dosage range described herein and the other pharmaceutically-active agent within its approved dosage range. Sequential use is contemplated when a combination formulation is inappropriate.
Although the compounds of the Formula I are primarily of value as therapeutic agents for use in warm-blooded animals (including man), they are also useful whenever it is required to inhibit the effects of cytokines. Thus, they are useful as pharmacological standards for use in the development of new biological tests and in the search for new pharmacological agents.
An amide derivative of the Formula I, or a pharmaceutically-acceptable salt or in-vivo-cleavable ester thereof, may be prepared by any process known to be applicable to the preparation of chemically-related compounds. Suitable processes are illustrated by, for example, those used in J. Med. Chem., 1996, 39, 3343-3356. Such processes, when used to prepare a novel amide derivative of the Formula I are provided as a further feature of the invention and are illustrated by the following representative process variants in which, unless otherwise stated, R1, R2, R3, R4, m, p and q have any of the meanings defined hereinbefore. Necessary starting materials may be obtained by standard procedures of organic chemistry. The preparation of such starting materials is described in conjunction with the following representative process variants and within the accompanying Examples. Alternatively necessary starting materials are obtainable by analogous procedures to those illustrated which are within the ordinary skill of an organic chemist.
a) A compound of the Formula I, or a pharmaceutically-acceptable salt or in-vivo-cleavable ester thereof, may be prepared by reacting an aniline of the Formula II 
xe2x80x83with an acid of the Formula III 
xe2x80x83or an activated derivative thereof, under standard amide bond forming conditions, wherein variable groups are as hereinbefore defined and wherein any functional group is protected, if necessary, and:
(i) removing any protecting groups;
(ii) optionally forming a pharmaceutically-acceptable salt or in-vivo-cleavable ester.
A suitable activated derivative of an acid of the Formula III is, for example, an acyl halide, for example an acyl chloride formed by the reaction of the acid and an inorganic acid chloride, for example thionyl chloride; a mixed anhydride, for example an anhydride formed by the reaction of the acid and a chloroformate such as isobutyl chloroformate; an active ester. for example an ester formed by the reaction of the acid with a phenol such as pentafluorophenol, with an ester such as pentafluorophenyl trifluoroacetate or with an alcohol such as N-hydroxybenzotriazole; an acyl azide, for example an azide formed by the reaction of the acid and an azide such as diphenylphosphoryl azide; an acyl cyanide, for example a cyanide formed by the reaction of an acid and a cyanide such as diethylphosphoryl cyanide; or the product of the reaction of the acid and a carbodiimide such as dicyclohexylcarbodiimide.
The reaction is preferably carried out in the presence of a suitable base such as, for example an alkali or alkaline earth metal carbonate, alkoxide, hydroxide or hydride, for example sodium carbonate, potassium carbonate, sodium ethoxide, potassium butoxide, sodium hydroxide, potassium hydroxide, sodium hydride or potassium hydride, or an organometallic base such as an alkyl-lithium, for example n-butyl-lithium, or a dialkylamino-lithium, for example lithium di-isopropylamide, or, for example, an organic amine base such as, for example, pyridine, 2,6-lutidine, collidine, 4-dimethylaminopyridine, triethylamine, morpholine or diazabicyclo[5.4.0]undec-7-ene. The reaction is also preferably carried out in a suitable inert solvent or diluent, for example tetrahydrofuran, 1,2-dimethoxyethane, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidin-2-one, dimethylsulphoxide or acetone, and at a temperature in the range, for example, xe2x88x9278xc2x0 to 150xc2x0 C., conveniently at or near ambient temperature.
Typically a carbodiimide coupling reagent is used in the presence of an organic solvent (preferably an anhydrous polar aprotic organic solvent) at a non-extreme temperature, for example in the region xe2x88x9210 to 40xc2x0 C., typically at ambient temperature of about 20xc2x0 C.
Protecting groups may in general be chosen from any of the groups described in the literature or known to the skilled chemist as appropriate for the protection of the group in question and may be introduced by conventional methods. Protecting groups may be removed by any convenient method as described in the literature or known to the skilled chemist as appropriate for the removal of the protecting group in question, such methods being chosen so as to effect removal of the protecting group with minimum disturbance of groups elsewhere in the molecule.
Specific examples of protecting groups are given below for the sake of convenience, in which xe2x80x9clowerxe2x80x9d, as in, for example, lower alkyl, signifies that the group to which it is applied preferably has 1-4 carbon atoms. It will be understood that these examples are not exhaustive. Where specific examples of methods for the removal of protecting groups are given below these are similarly not exhaustive. The use of protecting groups and methods of deprotection not specifically mentioned is of course within the scope of the invention.
A carboxy protecting group may be the residue of an ester-forming aliphatic or arylaliphatic alcohol or of an ester-forming silanol (the said alcohol or silanol preferably containing 1-20 carbon atoms).
Examples of carboxy protecting groups include straight or branched chain C 1-12alkyl groups (for example isopropyl, tert-butyl); lower alkoxy lower alkyl groups (for example methoxymethyl, ethoxymethyl, isobutoxymethyl); lower aliphatic acyloxy lower alkyl groups, (for example acetoxymethyl, propionyloxymethyl, butyryloxymethyl, pivaloyloxymethyl); lower alkoxycarbonyloxy lower alkyl groups (for example 1-methoxycarbonyloxyethyl, 1-ethoxycarbonyloxyethyl); aryl lower alkyl groups (for example benzyl, p-methoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, benzhydryl and phthalidyl); tri(lower alkyl)silyl groups (for example trimethylsilyl and tert-butyldimethylsilyl); tri(lower alkyl)silyl lower alkyl groups (for example trimethylsilylethyl), and C2-6alkenyl groups (for example allyl and vinylethyl).
Methods particularly appropriate for the removal of carboxyl protecting groups include for example acid-, base-, metal- or enzymically-catalysed hydrolysis.
Examples of hydroxy protecting groups include lower alkyl groups (for example tert-butyl), lower alkenyl groups (for example allyl); lower alkanoyl groups (for example acetyl); lower alkoxycarbonyl groups (for example tert-butoxycarbonyl); lower alkenyloxycarbonyl groups (for example allyloxycarbonyl); aryl lower alkoxycarbonyl groups (for example benzoyloxycarbonyl, p-methoxybenzyloxycarbonyl, o-nitrobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl); tri lower alkylsilyl (for example trimethylsilyl, tert-butyldimethylsilyl) and aryl lower alkyl (for example benzyl) groups.
Examples of amino protecting groups include formyl, aralkyl groups (for example benzyl and substituted benzyl, p-methoxybenzyl, nitrobenzyl and 2,4-dimethoxybenzyl, and triphenylmethyl); di-p-anisylmethyl and furylmethyl groups; lower alkoxycarbonyl (for example tert-butoxycarbonyl); lower alkenyloxycarbonyl (for example allyloxycarbonyl); aryl lower alkoxycarbonyl groups (for example benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, o-nitrobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl; trialkylsilyl (for example trimethylsilyl and tert-butyldimethylsilyl); alkylidene (for example methylidene); benzylidene and substituted benzylidene groups.
Methods appropriate for removal of hydroxy and amino protecting groups include, for example, acid-, base-, metal- or enzymically-catalysed hydrolysis for groups such as p-nitrobenzyloxycarbonyl, hydrogenation for groups such as benzyl and photolytically for groups such as o-nitrobenzyloxycarbonyl.
The reader is referred to Advanced Organic Chemistry, 4th Edition, by Jerry March, published by John Wiley and Sons 1992, for general guidance on reaction conditions and reagents. The reader is referred to Protective Groups in Organic Synthesis, 2nd Edition, by Green et al., published by John Wiley and Sons for general guidance on protecting groups.
The aniline of Formula II may be prepared by reduction of the corresponding nitro compound of Formula IV. 
Typical reaction conditions include the use of ammonium formate in the presence of a catalyst (for example palladium-on-carbon) in the presence of an organic solvent (preferably a polar protic solvent), preferably with heating, for example to about 60xc2x0 C. Any functional groups are protected and deprotected as necessary.
The compound of Formula IV may be prepared by reaction of an acid of Formula V, or an activated derivative thereof, 
with an aniline for Formula VI under suitable amide bond forming conditions. 
Typical conditions include activating the carboxy group of the compound of Formula V for example by treatment with a halo reagent (for example oxalyl chloride) to form an acyl halide in an organic solvent at ambient temperature, then reacting the activated compound with the aniline of Formula VI. Any functional groups are protected and deprotected as necessary.
(b) A compound of the Formula I, or a pharmaceutically-acceptable salt or in-vivo-cleavable ester thereof, may be prepared by reacting an acid of the Formula V 
xe2x80x83or an activated derivative thereof as defined hereinbefore, with an aniline of the Formula VII 
xe2x80x83under standard amide bond forming conditions, wherein variable groups are as hereinbefore defined and wherein any functional group is protected, if necessary, and:
(i) removing any protecting groups;
(ii) optionally forming a pharmaceutically-acceptable salt or in-vivo-cleavable ester.
The aniline of Formula VII may be prepared by reduction of the corresponding nitro compound using convention procedures as defined hereinbefore or as illustrated in the Examples.
(c) A compound of the Formula I wherein R1 or a substituent on R4 is an amino group may be prepared by the reduction of a compound of the Formula I wherein R1 or a substituent on R4 is a nitro group.
Typical reaction conditions include the use of ammonium formate or hydrogen gas in the presence of a catalyst, for example a metallic catalyst such as palladium-on-carbon. Alternatively a dissolving metal reduction may be carried out, for example using iron in the presence of an acid, for example an inorganic or organic acid such as hydrochloric. hydrobromic, sulphuric or acetic acid. The reaction is conveniently carried out in the presence of an organic solvent (preferably a polar protic solvent) and preferably with heating for example to about 60xc2x0 C. Any functional groups are protected and deprotected as necessary.
The following biological assays and Examples serve to illustrate the present invention.
Biological Assays
The following assays can be used to measure the p38 kinase-inhibitory, the TNF-inhibitory and anti-arthritic effects of the compounds of the present invention:
In vitro Enzyme Assay
The ability of compounds of the invention to inhibit the enzyme p38 kinase was assessed. Activity of particular test compounds against each of the p38xcex1 and p38xcex2 isoforms of the enzyme was determined.
Human recombinant MKK6 (GenBank Accesion Number G1209672) was isolated from Image clone 45578 (Genomics, 1996, 33, 151) and utilised to produce protein in the form of a GST fusion protein in a pGEX vector using analogous procedures to those disclosed by J. Han et al., Journal of Biological Chemistry, 1996, 271, 2886-2891. p38xcex1 (GenBank Accession Number G529039) and p38xcex2 (GenBank Accession Number G1469305) were isolated by PCR amplification of human lymphoblastoid cDNA (GenBank Accession Number GM1416) and human foetal brain cDNA [synthesised from mRNA (Clontech, catalogue no. 6525-1) using a Gibco superscript cDNA synthesis kit] respectively using oligonucleotides designed for the 5xe2x80x2 and 3xe2x80x2 ends of the human p38xcex1 and p38xcex2 genes using analogous procedures to those described by J.Han et al., Biochimica et Biophyhsica Acta, 1995, 1265, 224-227 and Y. Jiang et al., Journal of Biological Chemistry, 1996, 271, 17920-17926.
Both p38 protein isoforms were expressed in e coli in PET vectors. Human recombinant p38xcex1 and p38xcex2 isoforms were produced as 5xe2x80x2 c-myc, 6His tagged proteins. Both MKK6 and the p38 proteins were purified using standard protocols: the GST MKK6 was purified using a glutathione sepharose column and the p38 proteins were purified using nickel chelate columns.
The p38 enzymes were activated prior to use by incubation with MKK6 for 3 hours at 30xc2x0 C. The unactivated coli-expressed MKK6 retained sufficient activity to fully activate both isoforms of p38. The activation incubate comprised p38xcex1 (10 xcexcl of 10 mg/ml) or p38xcex2 (10 xcexcl of 5 mg/ml) together with MKK6 (10 xcexcl of 1 mg/ml), xe2x80x98Kinase bufferxe2x80x99 [100 xcexcl; pH 7.4 buffer comprising Tris (50 mM), EGTA (0.1 mM), sodium orthovanadate (0.1 mM) and xcex2-mercaptoethanol (0.1%)] and MgATP (30 xcexcl of 50 mM Mg(OCOCH3)2 and 0.5 mM ATP). This produced enough activated p38 enzyme for 3 Microtiter plates.
Test compounds were solubilised in DMSO and 10 xcexcl of a 1:10 diluted sample in xe2x80x98Kinase Bufferxe2x80x99 was added to a well in a Microtiter plate. For single dose testing, the compounds were tested at 10 xcexcM. xe2x80x98Kinase Assay Mixxe2x80x99 [30 xcexcl; comprising Myelin Basic Protein (Gibco BRL cat. no. 1322B-010; 1 ml of a 3.33 mg/ml solution in water), activated p38 enzyme (50 xcexcl) and xe2x80x98Kinase Bufferxe2x80x99 (2 ml)) was then added followed by xe2x80x98Labelled ATPxe2x80x99 [10 xcexcl; comprising 50 xcexcM ATP, 0.1 xcexcCi33P ATP (Amersham International cat. no. BF1000) and 50 mM Mg(OCOCH3)2]. The plates were incubated at room temperature with gentle agitation. Plates containing p38xcex1 were incubated for 90 min and plates containing p38xcex2 were incubated for 45 min. Incubation was stopped by the addition of 50 xcexcl of 20% trichloroacetic acid (TCA). The precipitated protein was phosphorylated by p38 kinase and test compounds were assessed for their ability to inhibit this phosphorylation. The plates were filtered using a Canberra Packard Unifilter and washed with 2% TCA, dried overnight and counted on a Top Count scintillation counter.
Test compounds were tested initially at a single dose and active compounds were retested to allow IC50 values to be determined.
In vitro Cell-based Assays
(i) PBMC
The ability of compounds of this invention to inhibit TNFxcex1 production was assessed by using human peripheral blood mononuclear cells which synthesise and secrete TNFxcex1 when stimulated with lipopolysaccharide.
Peripheral blood mononuclear cells (PBMC) were isolated from heparinised (10 units/ml heparin) human blood by density centrifugation (Lymphoprep(trademark); Nycomed). Mononuclear cells were resuspended in culture medium [RPMI 1640 medium (Gibco) supplemented with 50 units/ml penicillin, 50 xcexcg/ml streptomycin, 2 mM glutamine and 1% heat-inactivated human AB serum (Sigma H-1513)]. Compounds were solubilised in DMSO at a concentration of 50 mM, diluted 1:100 in culture medium and subsequently serial dilutions were made in culture medium containing 1% DMSO. PBMCs (2.4xc3x97105 cells in 160 xcexcl culture medium) were incubated with 20 xcexcl of varying concentrations of test compound (triplicate cultures) or 20 xcexcl culture medium containing 1% DMSO (control wells) for 30 minutes at 37xc2x0 C. in a humidified (5% CO2/95% air) incubator (Falcon 3072 ; 96 well flat-bottom tissue culture plates). 20 xcexcl lipopolysaccharide [LPS E.Coli 0111:B4 (Sigma L-4130), final concentration 10 xcexcg/ml] solubilised in culture medium was added to appropriate wells. 20 xcexcl culture medium was added to xe2x80x9cmedium alonexe2x80x9d control wells. Six xe2x80x9cLPS alonexe2x80x9d and four xe2x80x9cmedium alonexe2x80x9d controls were included on each 96 well plate. Varying concentrations of a known TNFxcex1 inhibitor were included in each test, i.e. an inhibitor of the PDE Type IV enzyme (for example see Semmler, J. Wachtel. H and Endres, S., Int. J. Immunopharmac. (1993), 15(3), 409-413) or an inhibitor of proTNFxcex1 convertase (for example, see McGeehan, G. M. et al. Nature (1994) 370, 558-561). Plates were incubated for 7 hours at 37xc2x0 C. (humidified incubator) after which 100 xcexcl of the supernatant was removed from each well and stored at xe2x88x9270xc2x0 C. (96 well round-bottom plates; Corning 25850). TNFxcex1 levels were determined in each sample using a human TNFxcex1 ELISA (see WO92/10190 and Current Protocols in Molecular Biology, vol 2 by Frederick M. Ausbel et al., John Wiley and Sons Inc.).
% inhibition=(LPS alonexe2x88x92medium alone)xe2x88x92(test concentrationxe2x88x92medium alone)xc3x97100 (LPS alonexe2x88x92medium alone)
(ii) Human Whole Blood
The ability of the compounds of this invention to inhibit TNFxcex1 production was also assessed in a human whole blood assay. Human whole blood secretes TNFxcex1 when stimulated with LPS. This property of blood forms the basis of an assay which is used as a secondary test for compounds which profile as active in the PBMC test.
Heparinised (10 units/ml) human blood was obtained from volunteers. 160 xcexcl whole blood were added to 96 well round-bottom plates (Corning 25850). Compounds were solubilised and serially diluted in RPMI 1640 medium (Gibco) supplemented with 50 units/ml penicillin. 50 xcexcg/ml streptomycin and 2 mM glutamine, as detailed above. 20 xcexcl of each test concentration was added to appropriate wells (triplicate cultures). 20 xcexcl of RPMI 1640 medium supplemented with antibiotics and glutamine was added to control wells. Plates were incubated for 30 minutes at 37xc2x0 C. (humidified incubator), prior to addition of 20 xcexcl LPS (final concentration 10 xcexcg/ml). RPMI 1640 medium was added to control wells. Six xe2x80x9cLPS alonexe2x80x9d and four xe2x80x9cmedium alonexe2x80x9d controls were included on each plate. A known TNFxcex1 synthesis/secretion inhibitor was included in each test. Plates were incubated for 6 hours at 37xc2x0 C. (humidified incubator). Plates were centrifuged (2000 rpm for 10 minutes) and 100 xcexcl plasma removed and stored at xe2x88x9270xc2x0 C. (Corning 25850 plates). TNFxcex1 levels were measured by ELISA (see WO92/10190 and Current Protocols in Molecular Biology, vol 2 by Frederick M. Ausbel et al., John Wiley and Sons Inc.). The paired antibodies that were used in the ELIZA were obtained from RandD Systems (catalogue nos. MAB610 anti-human TNFxcex1 coating antibody, BAF210 biotinylated anti-human TNFxcex1 detect antibody).
Ex vivo/In vivo Assessment
The ability of the compounds of this invention as ex vivo TNFxcex1 inhibitors were assessed in the rat or mouse. Briefly, groups of male Wistar Alderley Park (AP) rats (180-210 g) were dosed with compound (6 rats) or drug vehicle (10 rats) by the appropriate route. for example peroral (p.o.), intraperitoneal (i.p.) or subcutaneous (s.c.). Ninety minutes later rats were sacrificed using a rising concentration of CO2 and bled out via the posterior vena cavae into 5 Units of sodium heparin/ml blood. Blood samples were immediately placed on ice and centrifuged at 2000 rpm for 10 min at 4xc2x0 C. and the harvested plasmas frozen at xe2x88x9220xc2x0 C. for subsequent assay of their effect on TNFxcex1 production by LPS-stimulated human blood. The rat plasma samples were thawed and 175 xcexcl of each sample was added to a set format pattern in a 96 well round bottom plate (Corning 25850). 50 xcexcl of heparinized human blood was then added to each well, mixed and the plate was incubated for 30 min at 37xc2x0 C. (humidified incubator). LPS (25 xcexcl; final concentration 10 xcexcg/ml) was added to the wells and incubation continued for a further 5.5 hours. Control wells were incubated with 25 xcexcl of medium alone. Plates were then centrifuged for 10 min at 2000 rpm and 200 xcexcl of the supernatants were transferred to a 96 well plate and frozen at xe2x88x9220xc2x0 C. for subsequent analysis of TNF concentration by ELISA.
Data analysis by dedicated software calculates for each compound/dose:
% inhibition of TNFxcex1=Mean TNFxcex1 (Controls)xe2x88x92Mean TNFxcex1 (Treated)xc3x97100 Mean TNFxcex1 (Controls)
Alternatively, mice could be used instead of rats in the above procedure.
Test as Anti-arthritic Agent
Activity of a compound as an anti-arthritic agent was tested as follows. Acid soluble native type II collagen was shown by Trentham et al. [1] to be arthritogenic in rats; it caused polyarthritis when administered in Freunds incomplete adjuvant. This is now known as collagen-induced arthritis (CIA) and similar conditions can be induced in mice and primates. Recent studies have shown that anti-TNF monoclonal antibodies [2] and TNF receptor-IgG fusion proteins [3] ameliorate established CIA indicating that TNF plays a key role in the pathophysiology of CIA. Moreover, the remarkable efficacy reported for anti-TNF monoclonal antibodies in recent rheumatoid arthritis clinical trials indicates that TNF plays a major role in this chronic inflammatory disease. Thus CIA in DBA/1 mice as described in references 2 and 3 is a tertiary model which can be used to demonstrate the anti-arthritic activity of a compound. Also see reference 4.
1. Trentham, D. E. et al., (1977) J. Exp. Med., 146, 857.
2. Williams, R. O. et al., (1992) Proc. Natl. Acad. Sci., 89,9784.
3. Williams. R. O. et al., (1995) Immunology, 84, 433.
4 Badger, M. B. et al., (1996) The Journal of Pharmacology and Experimental Therapeutics, 279, 1453-1461.
Although the pharmacological properties of the compounds of the Formula I vary with structural change as expected, in general a compound of the Formula I gives over 30% inhibition in the PBMC test at concentrations up to 50 xcexcM. No physiologically unacceptable toxicity was observed at the effective dose for compounds tested of the present invention.
The invention will now be illustrated in the following non-limiting Examples in which, unless otherwise stated:
(i) operations were carried out at ambient temperature, i.e. in the range 17 to 25xc2x0 C. and under an atmosphere of an inert gas such as argon unless otherwise stated;
(ii) evaporations were carried out by rotary evaporation in vacuo and work-up procedures were carried out after removal of residual solids by filtration;
(iii) column chromatography (by the flash procedure) and medium pressure liquid chromatography (MPLC) were performed on Merck Kieselgel silica (Art. 9385) or Merck Lichroprep RP-18 (Art. 9303) reversed-phase silica obtained from E. Merck, Darmstadt, Germany or high pressure liquid chromatography (HPLC) was performed on C18 reverse phase silica, for example on a Dynamax C-18 60 xc3x85 preparative reversed-phase column;
(iv) yields are given for illustration only and are not necessarily the maximum attainable;
(v) in general, the end-products of the Formula I have satisfactory microanalyses and their structures were confirmed by nuclear magnetic resonance (NMR) and/or mass spectral techniques; fast-atom bombardment (FAB) mass spectral data were obtained using a Platform spectrometer and, where appropriate, either positive ion data or negative ion data were collected; NMR chemical shift values were measured on the delta scale [proton magnetic resonance spectra were determined using a Varian Gemini 2000 spectrometer operating at a field strength of 300 MHz or a Bruker AM250 spectrometer operating at a field strength of 250 MHz]; the following abbreviations have been used: s, singlet; d, doublet; t, triplet; m, multiplet; br, broad;
(vi) intermediates were not generally fully characterised and purity was assessed by thin layer chromatographic, HPLC, infra-red (IR) and/or NMR analysis;
(vii) melting points are uncorrected and were determined using a Mettler SP62 automatic melting point apparatus or an oil-bath apparatus; melting points for the end-products of the Formula I were determined after crystallisation from a conventional organic solvent such as ethanol, methanol, acetone, ether or hexane, alone or in admixture; and
(viii) the following abbreviations have been used:
DMF N,N-dimethylformamide
HPLC high pressure liquid chromatograph
DMSO dimethylsulphoxide